Tonic inhibition of TRPV3 by Mg2+ in mouse epidermal keratinocytes.

Abstract

The transient receptor potential vanilloid 3 channel (TRPV3) is abundantly expressed in epidermal keratinocytes and has important roles in sensory biology and skin health. Mg(2+) deficiency causes skin disorders under certain pathological conditions such as type 2 diabetes mellitus. In this study, we investigated the effect of Mg(2+) on TRPV3 in primary epidermal keratinocytes. Extracellular Mg(2+) ([Mg(2+)](o)) inhibited TRPV3-mediated membrane current and calcium influx. TRPV3 activation induced a calcium signaling pathway culminating in activation of the cAMP response element binding. TRPV3 inhibition by [Mg(2+)](o), the TRPV3 blocker ruthenium red, or TRPV3 siRNA suppressed this response. In TRPV3-expressing Chinese hamster ovary cells, both extracellular and intracellular Mg(2+) inhibited TRPV3 single-channel conductance, but not open probability. Neutralization of an aspartic acid residue (D641) in the extracellular pore loop or two acidic residues (E679, E682) in the inner pore region significantly attenuated the inhibitory effect of extracellular or intracellular Mg(2+) on TRPV3-mediated signaling, respectively. Our findings suggest that epidermal TRPV3 is tonically inhibited by both extracellular and intracellular Mg(2+), which act on both sides of the channel pore loop. Mg(2+) deficiency may promote the function of TRPV3 and contribute to the pathogenesis of skin diseases.

Inhibition of TRPV3-mediated current by [Mg2+]o in cultured mouse keratinocytes

(a) Representative current traces in response to voltage ramp from -100 mV to +100 mV without (basal) and with the TRPV3 agonist cocktail in the absence (0 mM) and presence of 2 and 10 mM Mg2+. (b) Percentage of inhibition of TRPV3 agonist cocktail-activated current by 10 mM Mg2+ at holding potential of -100 mV and +100 mV obtained from the voltage ramp shown in (a). Please note the different inhibitory effects at -100 mV and +100 mV. (c) Rectification ratio [-(I100/I-100)] with different concentrations of Mg2+ in the intracellular and extracellular solutions. ** p<0.01 compared with 2 mM Mg2+ in the extracellular solution with 2 or 10 mM Mg2+ in the intracellular solution. ## p<0.01 compared with 2 mM Mg2+ in the intracellular solution with 0, 2, or 10 mM Mg2+ in the extracellular solution.

Effect of [Mg2+]o on the I-V relationship of TRPV3-mediated single-channel current

(a) Representative single-channel current traces activated by 10 μM 2APB at holding potentials of +60 and -60 mV in the absence and presence of 3 mM Mg2+ in the extracellular solution in CHO cells expressing mTRPV3. (b) I-V relationships of the TRPV3 single-channel current activated by 2APB in CHO cells expressing mTRPV3. Current amplitudes at different holding potentials are normalized to that at -60 mV in the presence of 3 mM Mg2+ in the extracellular solution (marked as asterisk).

Effect of [Mg2+]i on the I-V relationship of TRPV3-mediated single-channel current

(a) Representative single-channel current traces activated by 10 μM 2APB at holding potentials of +60 and -60 mV in the absence and presence of 1 and 3 mM Mg2+ in the intracellular solution in CHO cells expressing mTRPV3. (b) I-V relationships of the TRPV3 single-channel current activated by 2APB in CHO cells expressing mTRPV3. Current amplitudes at different holding potentials are normalized to those at -60 mV in the presence of 3 mM Mg2+ in the intracellular solution (asterisk).

(a) Bar graph illustrating the inhibitory effect of 3 mM [Mg2+]o on TRPV3 mediated single-channel current amplitude with D641N mutation at holding potentials of -60 and −60 mV. (bi) Bar graph illustrating the inhibitory effects of 3 mM [Mg2+]i on wild-type and TRPV3 mutants with neutralizing mutaitons of acidic residues on the intracellular side near the channel pore. (bii) Graph illustrating the concentration-dependent inhibition of [Mg2+]i on the wild-type and TRPV3 mutants at concentrations ranging from 0 to 3 mM.

(a) Averaged response of 200 cells in time-lapse images from a representative coverslip illustrating the inhibitory effect of 10 mM Mg2+ on TRPV3 agonist cocktail-induced increase of [Ca2+]i. Horizontal bars indicate the time course of chemical applications. (b) Red bars are summarized data illustrating the responses to four consecutive applications of TRPV3 agonist cocktail in the absence (first and fourth response) and presence of 2 mM (second response) and 10 mM (third response) Mg2+. All responses are normalized to the first response. Black bars serve as non-treatment control showing the four consecutive responses without Mg2+ application in the control group.

Suppression of TRPV3-mediated increase of CREB transcriptional activity by Mg2+

(a) The CRE-Luc activity in control groups is set to 100% for a and b, and treated groups are normalized to this value (mean ± s.e.m. from 15 animals). Summarized data showing TRPV3 agonist cocktail-induced CRE-Luc activity in the absence or presence of ruthenium red (RR) in transgenic mouse keratinocytes as well as transgenic mouse keratinocytes transfected with TRPV3 siRNA and control siRNA. * p<0.05 compared with control; # p < 0.05. (b) TRPV3 agonist cocktail-induced CRE-Luc activity in the absence or presence or different concentrations of Mg2+ in keratinocytes from transgenic mice. * p<0.05.